Abstract
Huanglongbing (HLB) is the most devastating citrus disease and is associated with three bacterial species of the genus ‘Candidatus Liberibacter’ transmitted by insect vectors. The early detection of HLB is based on PCR methods, and it is one of the cornerstones for preventing incursion into disease-free countries. However, the detection of phytopathogenic bacteria with PCR-based methods is problematic in surveys that include a variety of samples of different origins. Here, we first report the proportion of amplifications obtained by two standardized real-time PCR methods for the diagnosis of HLB in various environmental samples that include plants, psyllid vectors, and parasitic wasps of the psyllids. The results of 4915 samples showed that 9.3% of them were amplified by the first rapid screening test and only 0.3% by the more specific tests. Most of the amplifications were associated with parasitic wasps. We designed the primers external to the target regions of both real-time PCR protocols to determine if amplifications belonged to one of three ‘Ca. Liberibacter’ species associated with HLB. The bioinformatic analysis of the sequences obtained with these primers revealed that all these amplifications came from the presence of other prokaryotic organisms in the samples. The primers developed in this study overcome the problem of undesired amplification in environmental samples. Thus, they could be used in future survey protocols to prevent the eradication of negative trees and the generation of unjustified alarms.
Highlights
Introduction in published maps and institutionalAccurate detection is one of the benchmarks of plant bacterial disease management.The challenge of detection is greater when the target organism cannot be grown under in vitro conditions, and detection is based only on molecular methods
Knowledge of how a real-time PCR protocol performs in routine analysis will permit its adequate integration into diagnostic schemes, with the correct interpretation of results, and the design of optimal risk management strategies [41]
Sensitivity, and specificity, speed, economy-sustainability, and ease of use are the main characteristics that a detection protocol must meet and knowing the advantages and drawbacks of those used in large-scale surveys is necessary
Summary
Introduction in published maps and institutionalAccurate detection is one of the benchmarks of plant bacterial disease management.The challenge of detection is greater when the target organism cannot be grown under in vitro conditions, and detection is based only on molecular methods. PCR-based methods have become essential in detection protocols; they present problems of sensitivity, specificity and robustness in complex environmental samples such as leaves, roots, insects and soil [1]. Sometimes it is necessary, after designing new primers, to adjust sequences, reagents and amplification conditions to increase the specificity of the reaction as new information becomes available [2]. HLB is associated with three Gram-negative species of bacteria restricted to phloem sieve tubes. These bacteria cannot be grown in the laboratory in pure culture at present
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